journal club presentation 2.pptx

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Journal Club Presentation-2
Presented by
R. Jyothsna Naidu
19L81S0104
II year, II semester
M. Pharmacy
Department of Pharmacology

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Title, Author and Affiliations
Impact factor: 3.3

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Literature
The above paper was chosen as it promotes my research study on Neurobehavioral
effects of probiotics feeding to young rats
Literature search were done from PubMed data base, Nature Scientific
Reports, Elsevier journals.
Journals were screened based on impact factor
and Science Citation Indexed (SCI), well peer
reviewed.
Recent research articles from 2015-2019
were screened
The following article is chosen after the
above justifications
Review articles were
excluded and research
articles were included

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Background- Gut Dysbiosis
• Gut microbiota consists of around 100 trillion microbial cells that provide the host with a broad variety of
metabolic functions.
• Human gut microbiome physiologically conducts variety of functions, including digestion, the maintenance
of immune and inflammatory homeostasis and the nervous system through the microbiome-gut-brain axis.
• Alteration in gut microbiome is Dysbiosis – cause for progression of many pathologies of the disorder.
• Recent studies have suggested that the gut microbiota could affect the behaviour of hosts and implicated their
involvement in depression, anxiety like behaviours, and modulating HPA axis
• A role for gut microbiota in the long-known link between diet and emotion has emerged over the past few
years, including evidence that high-fat diet feeding promotes a “leaky gut” (i.e., increased intestinal
permeability) similar to the effect of chronic stress alone , and the combination of high-fat diet and stress
exposure may promote even greater bacterial translocation.
• Increased circulating cytokines subsequent to bacterial translocation can sensitize the HPA axis to stress-
induced activation, and also can increase anxiety- and depressive-like behaviours.

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Background- Antibiotics
• The composition of gut microbiota is constantly affected by antibiotics, which are routinely
administered to treat various infections
• Clinical research has shown that antibiotics induce diarrhoea in children and older patients, and could
even cause IBD.
• These antibiotic-induced changes lead to immunity disorder, and increases susceptibility to infection
and impair the host immune system
• The hypothalamic-pituitary-adrenal axis (interaction between the hypothalamus, pituitary gland and
adrenal gland)- modulates the immune response, with high levels of cortisol resulting in a suppression
of immune and inflammatory reactions. This help to protect the organism from a lethal overactivation
of the immune system and minimizes the tissue damage from inflammation.
• The gut-brain axis, this bidirectional communication network - potential target for novel
antidepressant treatments.

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Background- Probiotics
• Probiotics are a combination of live beneficial bacteria and/or yeasts that naturally live
in your body
• Probiotics are made up of good bacteria that helps keep your body healthy and
working well.
• Probiotic biotherapies are known to create a healthy gut environment by balancing
bacterial populations and promoting their favorable metabolic action.
• “Psychobiotics” - a live organ-ism that produces a health benefit in patients suffering
from a psychiatric illness.
• When applied in adequate amounts probiotics are beneficial to host; they improve
colonic barrier function impaired by early life stress and have been used to treat many
illnesses such as diarrhoea, IBD and other gut disorders.

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Aim and Objectives
Aim
To evaluate the protective effect of Lactobacillus fermentum strain NS9 on Ampicillin induced anxiety like behaviour and hippocampal dependent spatial
memory in rats.
Objectives
 To feed the rats with ampicillin (120mg/kg) and Lactobacillus fermentum strain NS9 (109 cfu/ml)
 To perform behavioral parameters like Elevated plus maze, Morris water maze .
 To assess the Myeloperoxidase activity in the rat colon
 To measure the brain derived neurotrophic factor (BDNF), N-methyl- D-aspartate (NMDA), glucocorticoid receptor (GR) and mineralocorticoid receptor
(MR) levels were measured with commercial ELISA kits
 To assess the HPA axis activity by measuring the corticosterone level in rat serum.
 To perform qPCR

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Details of the Study
• Animals required: Thirty male Sprague-Dawley rats (10 rats per group)
• Treatment: L. fermentum strain NS9 and Ampicillin 120mg/kg
• Probiotic information: L. fermentum strain NS9 (laboratory designated numbers) was isolated from naturally fermented dairy products collected from
grasslands in Inner Mongolia, China P.R. The genome of this strain was sequenced (1,502 bp) and given the following unique GenBank accession no.
JQ013298.1.
The NS9 strain was incubated in De Man, Rogosa and Sharpe (MRS) broth at 37 °C overnight, collected by centrifugation at 3000 rpm for 10 min, and
washed twice in saline (0.9% NaCl). The pellet was then re-suspended in sterile water and added to the drinking water for rats at a concentration of 109
cfu/ml. This water was provided to the rats and was changed every day.
• Duration of the study: 41 days
• Diet- ad libitum with tap water
All animal experimentation was performed according to the guidelines for the care and use of laboratory animals approved by Institute of Psychology, Chinese Academy of Sciences. Thirty weaned male Sprague-Dawley rats (Rattus norvegicus) were purchased from the Vital River
Laboratories Co., Ltd. (Beijing, China P.R.).

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Study design and Outline

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Methods- Behavioural Parameters
• In the elevated maze the total distance travelled and the number of arm entries was recorded as an index of
general locomotor activity.
• The percentage of open arm entries (defined as four paws inside the maze arm) was quantified as a measure
of anxiety-like behaviour.
Elevated Plus Maze
• Animals had 4 trials per day (separated by 15 mins) for four consecutive days (day 32 to 35)- Phase 1 (SW
quadrant)
• A probe trial was performed on the day 36
• Phase 2 started on day 37- NE quadrant
• The rats were trained for 2 days from day 37 to 38
• On day 39 and 41 the platform was removed and the tests were carried out.
• The swimming speed, distance, latency to the platform and the time spent in each quadrant were recorded
Morris Water Maze

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Other Measures Estimated
qPCR
Gut microbiome
composition
Myelope
roxidase
activity
Colon
inflammation
Serum
corticost
erone
BDNF,NMDA,
MR, GR

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AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Statistical analysis
Results are expressed as mean ± standard error (SE). Nonparametric statistics (Mann Whitney U-test) was used
to analyze the gut microbiota data. In other tests, groups were compared by analysis of variance (ANOVA)
using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Post hoc analysis (Bonferroni or Student-Newman-Leuls)
were performed, as indicated. P-values of <0.05 and <0.01 were considered significant and extremely
significant, respectively.

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Results
• qPCR
Observed similar levels of Bacteroides, C. coccoides, Firmicutes and Lactobacillus in all three groups before ampicillin
or L. fermentum NS9 administration. (p>0.05)
Amp- led to decrease in Bacteroides and Lactobacillus and in increase C. coccoides, Firmicutes when compared to
control group (p<0.01)
Amp+ NS9- Bacteroides was similar to the control rats (p>0.05), the level of C. coccoides and Firmicutes was reduced
significantly when compared to ampicillin treated rats (p<0.01), NS9 application also increased the level of Lactobacillus
by 20%, which were significantly higher than control rats and ampicillin treated rats (p<0.01).
Mann Whitney U-test analysis
A- The composition of gut microbiota in rat faeces before treatment with Amp and NS9
B- The composition of gut microbiota in rat faeces at the end of the experiment
*p<0.05, compared to the control
#p<0.05 significant differences between the groups.

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SIRO- DSIR

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Results
• Elevated Plus Maze
1. Compared to controlled rats, ampicillin treated rats showed an obvious decrease in the
percentage of open-arm entries and increase in the closed arm entries.
2. Treatment with L. fermentum NS9 alleviated this abnormal behaviour and elevated the
percentage of open arm entries and reduce the percentage of close arm entries (p<0.05).
A- Total distance travelled
B- number of total arm entries
C- percentage open arm entries
D- percentage of closed arm entries
*p<0.05, compared to control group rats
#p<0.05, comparison between groups
One-way ANOVA followed by post-hoc Bonferroni test.

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Results
• Morris Water Maze
Phase 1:Day 32-36; target quadrant: SW- platform is located
1. The control group exhibited shorter latency than the Amp and Amp+NS9 on the first day of training.
2. The difference between the three groups gradually diminished over time.
3. Until the 4th day, the three groups had similar latency to find the platform.
4. On the 5th day (treatment day 36), platform was removed in test 1, all rats in three groups spent a similar amount of time in the platform.
A- Escape latency to find platform in experiment 1. Each block represents mean of four trials. *p<0.05, compared to the control group
B- Percentage of time spent in the 4 quadrants *p<0.05, the target quadrant compared to other quadrants
Phase 2:
1.The platform was removed, to the opposite quadrant (NE) to allow the rats to learn new position of the platform.
2. Day 39- the platform was removed and the rats were subjected to 2nd probe test
3. Control and Amp+NS9 treated rats spent most of their time in 2 quadrants- the quadrant where the platform was previously located and the
new quadrant which contained the platform.
4. Ampicillin treated rats spent nearly equal times in all 4 quadrants.

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AUTONOMOUS
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SIRO- DSIR
Probe test 3 (after 2 days from Day 39 i.e., day 41)
• This was done to investigate the ampicillin effects on long term memory.
• Result was the ampicillin treated rats still spent equal times (25%) in the 4 quadrants.
• The control and NS9 treated spent (35 & 30% respectively) significantly more time in SW quadrant than amp treated rats
(p<0.05)
Rat performance in second phase of MWM
A- Latency to find platform in every trial during training stage, *p<0.05, compared to control group.
B- Percentage time spent in the 4 quadrants in probe test 2 days after 24h(day 39), *p<0.05, other quadrants versus the target
quadrant NE
C- Percentage time spent in the 4 quadrants in probe test 3 after 72 h (day 41) , *p<0.05, other quadrants versus the target quadrant
SW.

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AUTONOMOUS
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Results
• Myeloperoxidase activity
• Ampicillin treatment is known to induce colon inflammation.
• In contrast to control group (4.3±0.9 U/mg), colon tissue in the ampicillin treated group had higher
MPO activity ((6.7±1.1 U/mg, P<0.05) which reduced to the normal value (5.0±1.1 U/mg, P<0.05)
after L. fermentum NS9 treatment (P>0.05).
*p<0.05, compared to the control group.
• Effect of Lactobacillus fermentum on serum corticosterone
• Ampicillin application markedly elevated serum corticosterone levels (from 550 to710 ng/l).
• Treatment with L. fermentum NS9 restored corticosterone levels to similar levels as the control rats
*p<0.05, compared to the control group.

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AUTONOMOUS
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Results
• Effect of Lactobacillus fermentum on BDNF, NMDA, MR and GR levels
1. The level of BDNF in the hippocampus was not different the groups, neither ampicillin application or NS9
application changed the levels of BDNF in the hippocampus.
2. Two kinds of corticosterone receptors, MR and GR, have been reported in the hippocampus, which is the main
target of corticosterone.
3. Treatment with ampicillin and NS9 had no effect on GR levels. However, ampicillin administration significantly
reduced MR levels (p<0.05)
4. Such a decrease was not observed in the NS9 treated group, which had remarkably increased levels of MR, when
compared to control levels (p<0.05)
5. Ampicillin also significantly reduced the NMDA receptor levels from 1,249±299 to 835±127 ng/g tissue.
6. Treatment with NS9 inhibited the ampicillin induced NMDA receptor reduction and maintained it at the normal
level (1,192±176 ng/g tissue)
A- Brain derived neurotrophic factor (BDNF), B- Glucocorticoid receptor, C- Mineralocorticoid receptor, D- N-methyl-D-
Aspartate protein levels in rat hippocampus treated by ampicillin (Amp) and Lactobacillus fermentum NS9..
* P<0.05, compared to the control group
# P<0.05, differences between groups

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AUTONOMOUS
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Interpretation: Gut microbiota
• Treatment with ampicillin affected with gut physiology, as well as emotional and spatial memory behaviour of male SD rats.
• NS9 application ameliorated the colon inflammation and normalized the emotional behaviour and spatial memory deficits induced
by ampicillin.
• NS9 restored the serum corticosterone levels and the hippocampus MR and NMDA receptor levels in the normalized gut
microbiota.
• qPCR:
• Treatment with ampicillin induced marked changes in the intestinal microbiota composition as evidenced by a decrease in
Lactobacillus and Bacteroides levels and an increase in C. coccoides and Firmicutes in rat guts.
• NS9 application improved the composition of gut microbiota by reducing the level of C. coccoides and Firmicutes. NS9 application
also increased the level of Lactobacillus by 20%, which were significantly higher than control rats and ampicillin treated rats.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Interpretation:Elevated Plus Maze:
1.Ampicillin treatment did not change the locomotor activity, which was shown by the total
distance travelled and total arm entries by the rats but induced mild anxiety like behaviour.
2. Compared to control rats, ampicillin treated rats showed an obvious decrease in the
percentage of open-arm entries and increase in the closed-arm entries.
3. Treatment with L. fermentum NS9 alleviated this abnormal behaviour and elevated the
percentage of open arm entries and reduced the percentage of close arm entries.
4. There was no significant difference in the time spent in the open arm or closed arm between
the Amp and control groups (data not shown)

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AUTONOMOUS
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Phase 1 Phase 2
1. The control group exhibited shorter latency than the
Amp and the Amp+NS9 groups on the first day of
training. This demonstrated that ampicillin marginally
impaired the learning behavior, which was not
alleviated by NS9 application.
2. The difference between the 3 groups diminished over
time, until the 4th day the three groups had similar
latency to find the platform. On 5th day when the
platform was removed in test 1 all rats in the three
groups spent a similar amount of time in the platform,
indicating that all rats remembered the location of the
platform.
1. The platform was removed to opposite quadrant to
allow the rats to learn new position.
2. The latency of the control rats was shorter than the
amp treated rats and NS9 treatment didn’t reduce the
latency to the control level suggesting that learning
of rats fed on ampicillin was slower than the control
rats.
3. After 6 trials it was observed that rats in the three
groups had a similar latency to the escape platform
4. On day 39, the platform was removed and the rats
were subjected to the 2nd probe test, in which the
control and Amp+NS9 treated rats spent their most
of the time in two quadrants; the quadrant where the
platform was previously located and the new
quadrant which contained the platform. This
indicated that rats in these two groups remembered
the two locations of the escape platform.
5. The ampicillin treated rats spent nearly equal times
in all the 4 quadrants suggesting that ampicillin
treated rats did not only forget the new location but
also forget the previous location.
Interpretation (Morris Water Maze)

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• To investigate the ampicillin effects on long term memory, the probe test was repeated again after 2 days.
• There were significant differences among the three groups, the amp treated groups still spent equal time in 4
quadrants.
• The control and the NS9 treated rats spent more time in quadrant SW (the previous platform) than the
ampicillin treated rats.
• This result shows that NS9 application significantly alleviated the memory defects induced by ampicillin.
• Note: All rats in the three groups forgot the new location indicating that 6 trials were not enough to maintain the
location memory for 72h
Interpretation: Morris Water Maze

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Interpretation: Myeloperoxidase
• Here MPO activity is used as a measure to monitor the mucosal inflammation induced by
antibiotic treatment.
• The observed reduction in inflammation caused by NS9 application in the present study
may be associated with the restoration of gut microbiota by NS9.
• Also, this reduction is MPO could be at least in part is attributed to the alleviate anxiety-like
behaviour in NS9 treated rats.

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Interpretation: Serum corticosterone
• Elevated corticosterone levels associated with anxiety like behaviour and also important in
learning and memory.
• This was signified in the present study with increased corticosterone levels and anxiety like
behavior along with impaired spatial memory observed in ampicillin treated groups.
• Treatment with NS9 along with ampicillin resulted in diminished levels of corticosterone
and alleviated anxiety like behavior in animals indicating the effect NS9 on behavior was
at physiological level.

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Interpretation: BDNF, NMDA, MR and GR levels
• MR and GR are being the important target of corticosterone to bring out its action are concentrated in
hippocampus possessing a key role in learning and memory.
• Further reports suggest that central MR antagonism and loss of forebrain MR are linked to impair memory
and its overexpression caused enhanced memory and spatial memory retention.
• Ampicillin treatment resulted in declension of MR levels in the hippocampus and the rats failed to show
quadrant preference similar to rats in the other two groups, suggesting that ampicillin impaired memory by
reducing MR in the hippocampus.
• Treatment with NS9, resulted in recovery of quadrant preference and increased hippocampal MR levels.
Note: Ampicillin had no effect on GR levels and BDNF levels (not in agreement with previous reports)

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• The NMDA receptor is a ligand-gated ion channel of glutamate essential for long-term
potentiation and hippocampal-dependent spatial learning and memory.
• It has been reported that NMDA receptor interacts with MR and GR to modulate memory.
• Ampicillin induced memory impairment was partly caused by reduction in levels of
NMDA receptors along with MR.
• While the NS9 administration elevated the expression of both MR and NMDA receptors in
the study. Animals fed on NS9 showed an enhanced memory of the first platform location
compared to ampicillin

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• The lack effect of probiotic NS9 on BDNF, implies that probiotic effects are strain
dependent and could function in different ways.
• This suggest that the clinical use of probiotics must be performed with caution, and more
animal and clinical research is needed to address this issue.

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Authors conclusion
Authors exemplified that using even a single antibiotic ampicillin also could cause gut dysbiosis, as well as
emotional distress and spatial memory impairment. Administration of NS9 was evidenced to be beneficial
as it could restore ampicillin induced physiological and psychological abnormalities in rats. The data in the
present study provided a basis for further investigation of effectiveness of probiotics in the maintenance of
brain health and adjunctive treatment of adverse effects induced by clinical antibiotic application.

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Critical Analysis
Title: Lactobacillus fermentum NS9 restores the antibiotic induced physiological and psychological abnormalities in rats
The title was justified and the given results proved that Lactobacillus fermentum NS9 could alleviate physiological and
psychological abnormalities induced by antibiotic.
Background of research:
Well structured background on Gut microbiota, HPA axis, antibiotic consumption resulted physiological and psychological
via gut dysbiosis, altering, MPO corticosterone levels and its related receptors GR and MR (disrupted spatial memory) was
described in the study and supported the outcome of the results, which were in consistent with previous reports.
Aim and objectives:
All the objectives mentioned were evaluated and proper interpretation of data with statistical analysis were provided

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AUTONOMOUS
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Critical Analysis
Validity of methods
All validated methods were used for behavioral parameters and all the manufactures instructions were followed for the
biochemical assessments like MPO, corticosterone, MR, GR and BDNF levels measurement and qPCR.
Animals and diet
All animal experimentation was performed according to the guidelines for the care and use of laboratory animals approved
by Institute of Psychology, Chinese Academy of Sciences.
Treatment:
Appropriate critical guidelines were followed

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Results
Results of each parameter were interpreted with respective statistical analysis and p<0.05 and <0.01 were considered
significant and extremely significant respectively
Conclusion
Conclusions were to the point and justified the present study.
Author limitations:
• Young adult rats were included, gut microbiota changes with age and the effect of probiotics may thus be different in
older rats.
• Further, the measurement of the total levels of NMDA receptor and MR in the hippocampus, as the levels of these
receptors were not examined in the subfields of hippocampus.
• There was no data shown for time spent in the open arm or closed arm between the Amp and control groups.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Overview
• The authors in the study tried to unravel the pivotal role played by gut microbiota in maintaining the health of the host and affect of antibiotics on gut
microbiome as one of the major factors causing dysbiosis.
• Appertaining reports describing the microbiome-protective role probiotics applications led the authors to investigate how probiotics alter the
physiological and psychological changes induced by antibiotics.
• They tested the performance of ampicillin-treated rats in the presence or absence of Lactobacillus fermentum strain NS9 acting as probiotic in this
case.
• Initially, gut microbiota assessment was done to assess the effect of ampicillin on gut microbiome in presence and absence of NS9 was done.
• Followed by physiological assessments like estimation of MPO levels in colon, serum corticosterone and its receptor (MR and GR) levels along with
NMDA receptor levels were performed. While the elevated plus maze and Morris water maze were carried out to investigate the psychological
behavior of animals.

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AUTONOMOUS
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NBA (UG)
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• The results showed the use NS9 normalized the composition of gut microbiota and alleviated the ampicillin-induced
inflammation in the colon.
• The levels of the mineralocorticoid and N-methyl-D-aspartate receptors were also elevated in the hippocampus of the
ampillicin+NS9 treated group.
• NS9 administration also reduced the anxiety-like behaviour and alleviated the ampicillin-induced impairment in
memory retention.
• These findings concluded the suggest that NS9 is beneficial to the host, because it restores the physiological and
psychological abnormalities induced by ampicillin highlighting how gut contents regulate the brain, and shed light on
the clinical applications of probiotics to treat the side effect of antibiotics and mental disorders
Overview

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AUTONOMOUS
NAAC &
NBA (UG)
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Applicability of findings of article
• The concept of psychobiotics, bacteria with positive effects on mental health.
• To develop probiotic therapeutic strategies to tackle antibiotic induced physiological and psychological disorders based on microbiota-gut-brain
axis.
• To fill the gap between laboratory scientists studying the gut-brain connection and people suffering antibiotic induced gut dysbiosis with psychiatric
disorders and cognitive challenges
• These results emphasize the complex influence of probiotics on the long run adverse effects of antibiotics leading to impaired special memory and
aggravated inflammatory disorders through modulating microbiota-gut-brain axis and highlight the need for additional research to further unravel
complex interactions and causal relationships among augmented use of antibiotics, the microbial gut-brain axis and beneficial yet cautious use of
probiotics.
• Probiotic effects are strain dependent and could function in different ways.
• Previous studies have shown that administering a mixture of three antibiotics could change mice behaviour and central biochemical levels. Our
results in the present study suggested that even one antibiotic, which is considered as safe and have less adverse effects, could induce gut microbiota
dysbiosis and behaviour abnormality.

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